We have identified an analgesic mechanism of linaclotide: it activates GC-C expressed on mucosal epithelial cells, resulting in the production and release of cGMP. This extracellular cGMP acts on and inhibits nociceptors, thereby reducing nociception. We also found that linaclotide reduces chronic abdominal pain in patients with IBS-C.

The capacities of urinary trefoil factor 3 (TFF3) and urinary albumin to detect acute renal tubular injury have never been evaluated with sufficient statistical rigor to permit their use in regulated drug development instead of the current preclinical biomarkers serum creatinine (SCr) and blood urea nitrogen (BUN). Working with rats, we found that urinary TFF3 protein levels were markedly reduced, and urinary albumin were markedly increased in response to renal tubular injury. Urinary TFF3 levels did not respond to nonrenal toxicants, and urinary albumin faithfully reflected alterations in renal function. In situ hybridization localized TFF3 expression in tubules of the outer stripe of the outer medulla. Albumin outperformed either SCr or BUN for detecting kidney tubule injury and TFF3 augmented the potential of BUN and SCr to detect kidney damage. Use of urinary TFF3 and albumin will enable more sensitive and robust diagnosis of acute renal tubular injury than traditional biomarkers.

The Predictive Safety Testing Consortium's first regulatory submission to qualify kidney safety biomarkers revealed two deficiencies. To address the need for biomarkers that monitor recovery from agent-induced renal damage, we scored changes in the levels of urinary biomarkers in rats during recovery from renal injury induced by exposure to carbapenem A or gentamicin. All biomarkers responded to histologic tubular toxicities to varied degrees and with different kinetics. After a recovery period, all biomarkers returned to levels approaching those observed in uninjured animals. We next addressed the need for a serum biomarker that reflects general kidney function regardless of the exact site of renal injury. Our assay for serum cystatin C is more sensitive and specific than serum creatinine (SCr) or blood urea nitrogen (BUN) in monitoring generalized renal function after exposure of rats to eight nephrotoxicants and two hepatotoxicants. This sensitive serum biomarker will enable testing of renal function in animal studies that do not involve urine collection.

BackgroundArterial media calcification (AMC) is highly prevalent and is a major cause of morbidity, mortality, stroke and amputation in patients with diabetes mellitus (DM). Previous research suggests that advanced glycation end products (AGEs) are responsible for vascular calcification in diabetic patients. The potential link between oxidative stress and AGEs-induced vascular calcification, however, has not been examined.MethodsMale Wistar rats received a high fat diet for 8 weeks followed by a single dose of streptozotocin to induce DM (DM). Calcification was induced with Vitamin D3 and nicotine (VDN). We started VDN treatment at 1 week after the initial streptozotocin injection (DM+VDN). Age-matched rats were used as controls (CON). Metabolic parameters, aortic calcium content, alkaline phosphatase (ALP) protein, malondialdehyde (MDA) content, Cu/Zn superoxide dismutase (SOD) activity, aorta receptor for advanced glycation end products (RAGE) and aorta AGEs levels were measured. In vitro, vascular smooth muscle cells (VSMCs) were cultured with AGEs in DMEM containing 10 mmol·L-1 ß -glycerophosphate (ß-GP). Calcium content and ALP activity were used to identify osteoblastic differentiation and mineralization. Western blots were used to examine protein expression of Cu/Zn SOD, NADPH oxidase Nox1 and RAGE. In addition, the intracellular reactive oxygen species (ROS) generation was evaluated using fluorescent techniques with dihydroethidine (DHE) method.ResultsThe DM+VDN group showed a significant increase in aortic calcium content, levels of aorta AGEs, MDA content, ALP protein levels and RAGE expression, although Cu/Zn SOD activity decreased significantly. In vitro, enhanced Nox1, RAGE expression as well as the production of intracellular superoxide anions, and reduced expression of Cu/Zn SOD induced by AGEs were attenuated by the anti-RAGE antibody or a ROS inhibitor. Furthermore, the AGEs-stimulated ROS increase was also significantly inhibited by a SOD mimetic. Increased ALP activity and calcium deposition were also inhibited markedly by the ROS inhibitor and the anti-RAGE antibody.ConclusionsThese results suggest that AGEs enhance vascular calcification partly through a RAGE/oxidative stress pathway.

The NK homeodomain factor Tinman is a crucial regulator of early mesoderm patterning and, together with the GATA factor Pannier and the Dorsocross T-box factors, serves as one of the key cardiogenic factors during specification and differentiation of heart cells. Although the basic framework of regulatory interactions driving heart development has been worked out, only about a dozen genes involved in heart development have been designated as direct Tinman target genes to date, and detailed information about the functional architectures of their cardiac enhancers is lacking. We have used immunoprecipitation of chromatin (ChIP) from embryos at two different stages of early cardiogenesis to obtain a global overview of the sequences bound by Tinman in vivo and their linked genes. Our data from the analysis of ∼50 sequences with high Tinman occupancy show that the majority of such sequences act as enhancers in various mesodermal tissues in which Tinman is active. All of the dorsal mesodermal and cardiac enhancers, but not some of the others, require tinman function. The cardiac enhancers feature diverse arrangements of binding motifs for Tinman, Pannier, and Dorsocross. By employing these cardiac and non-cardiac enhancers in machine learning approaches, we identify a novel motif, termed CEE, as a classifier for cardiac enhancers. In vivo assays for the requirement of the binding motifs of Tinman, Pannier, and Dorsocross, as well as the CEE motifs in a set of cardiac enhancers, show that the Tinman sites are essential in all but one of the tested enhancers; although on occasion they can be functionally redundant with Dorsocross sites. The enhancers differ widely with respect to their requirement for Pannier, Dorsocross, and CEE sites, which we ascribe to their different position in the regulatory circuitry, their distinct temporal and spatial activities during cardiogenesis, and functional redundancies among different factor binding sites.

SUMMARYMembers of the T-Box gene family of transcription factors are important players in regulatory circuits that generate myogenic and cardiogenic lineage diversities in vertebrates. We show that during somatic myogenesis in Drosophila, the single ortholog of vertebrate Tbx1, optomotor-blind-related-gene-1 (org-1), is expressed in a small subset of muscle progenitors, founder cells and adult muscle precursors, where it overlaps with the products of the muscle identity genes ladybird (lb) and slouch (slou). In addition, org-1 is expressed in the lineage of the heart-associated alary muscles. org-1 null mutant embryos lack Lb and Slou expression within the muscle lineages that normally co-express org-1. As a consequence, the respective muscle fibers and adult muscle precursors are either severely malformed or missing, as are the alary muscles. To address the mechanisms that mediate these regulatory interactions between Org-1, Lb and Slou, we characterized distinct enhancers associated with somatic muscle expression of lb and slou. We demonstrate that these lineage-and stage-specific cis-regulatory modules (CRMs) bind Org-1 in vivo, respond to org-1 genetically and require T-box domain binding sites for their activation. In summary, we propose that org-1 is a common and direct upstream regulator of slou and lb in the developmental pathway of these two neighboring muscle lineages. Cross-repression between slou and lb and combinatorial activation of lineage-specific targets by Org-1-Slou and Org-1-Lb, respectively, then leads to the distinction between the two lineages. These findings provide new insights into the regulatory circuits that control the proper pattering of the larval somatic musculature in Drosophila.

Endotoxin is one of the principal components of grain dust that causes acute reversible airflow obstruction and airway inflammation. To determine whether endotoxin responsiveness influences the development of chronic grain dust-induced airway disease, physiological and airway inflammation remodeling parameters were evaluated after an 8-wk exposure to corn dust extract (CDE) and again after a 4-wk recovery period in a strain of mice sensitive to (C3H/HeBFeJ) and one resistant to (C3H/HeJ) endotoxin. After the CDE exposure, both strains of mice had equal airway hyperreactivity to a methacholine challenge; however, airway hyperreactivity persisted only in the C3H/HeBFeJ mice after the recovery period. Only the C3H/HeBFeJ mice showed significant inflammation of the lower airway after the 8-wk exposure to CDE. After the recovery period, this inflammatory response completely resolved. Lung stereological measurements indicate that an 8-wk exposure to CDE resulted in persistent expansion of the airway submucosal cross-sectional area only in the C3H/HeBFeJ mice. Collagen type III and an influx of cells into the subepithelial area participated in the expansion of the submucosa. Our findings demonstrate that subchronic inhalation of grain dust extract results in the development of chronic airway disease only in mice sensitive to endotoxin but not in mice that are genetically hyporesponsive to endotoxin, suggesting that endotoxin is important in the development of chronic airway disease.

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